Thyroxine determination method employing the competitive protein binding technique

ABSTRACT

A method for use in determination of thyroxine in biological fluids which comprises extracting thyroxine with tetrahydrofuran and separating thyroxine from the precipitated proteins and then from the tetrahydrofuran, and thereafter equilibrating with thyroxine binding globulin and radioactive labeled thyroxine, removing unbound thyroxine and determining the radioactive labeled thyroxine by counting techniques.

United States Patent 1 [111 3,730,684 Demetriou May 1, 1973 [s41 --THYROXINE DETERMINATION [56] References Cited METHOD EMPLOYING THE UNITED STATES PATENTS COMPETITIVE PROTEIN BINDING Inventor: James A. Demetriou, Sherman Oaks,

Calif.

Assignee: Bio-Science Laboratories, Van Nuys, Calif.

Filecli Nov. 1, 1971 Appl. No.: 194,683

US. c1; ..23'/230 B, 250/106 T Int. Cl ..G0ln 33/16, GOln 23/10 Field of Search ..23/23O B, 253; I r I 250/106 T, 44, 71.5

Primary ExaminerMorris O. Wolk Assistant Examiner-R. E. Scrwin Attorney-William M. Yates et a1.

[5 7] ABSTRACT thyroxine binding globulin and radioactive labeled thyroxine, removing unbound thyroxine and determining the radioactive labeled thyroxine by counting techniques.

5 ClaimgNo Drawings THYROXINE DETERMINATION METHOD EMPLOYING THE COMPETITIVE PROTEIN BINDING TECHNIQUE BACKGROUND OF THE INVENTION The determination of thyroxine in biological fluids using a competitive protein binding procedure is an accepted method in many commercial, reference, hospital and industrial clinical laboratories. The method is based on the equilibration of thyroxine separated from the biological fluid with radioactive labeled thyroxine and thyroxine binding globulin (TBG). After equilibration the labeled thyroxine displaced from the TBG is removed by adding a resin. Assay of the amount of labeled thyroxine bound to TBG is proportional to the amount of thyroxine present in the biological fluid.

Principles of the technique are'described, for example, by Murphy, U. S. Pat. No. 3,414,383, and Murphy and Pattee, J. Clin. Endocr. 24; 187 (1965).

In such procedures it is essential to separate the thyroxine in the sample to be tested from any binding proteins which may be present in the sample. Such separation ordinarily requires release of bound thyroxine from binding proteins and precipitation of protein followed by removal of protein from the sample. Many precipitating agents which are satisfactory in other analytical determinations are unsuitable for use in this step since they also remove thyroxine along with the protein. Lower alkanols and acetone-alcohol mixtures have been used with a considerable degree of success. However, recoveries of thyroxine have been less than completely satisfactory, generally between about 70 and 85 percent. This lower recovery generally results in a less precise measurement of thyroxine.

A high degree of care and manual dexterity has been necessary in the addition of the alcoholic precipitants to the samples and in careful mixingof the precipitant and sample in order to avoid decreased recoveries. These steps are time-consuming and difficult to adapt to operations such as large clinical or hospital laboratories which must handle'agreat number of analyses each day.

SUMMARY OF THE INVENTION It has now been found that thyroxine recoveries in thyroxine assays by competitive protein binding can be greatly increased, and the time-consuming, difficult operations of protein precipitation can be greatly simplified by the use of tetrahydrofuran rather than alkanols, to release thyroxine and precipitate protein and afford a 9,4 to 98 percent recovery of the thyroxine from the serum samples.

In the improved process of the invention, the determination of thyroxine by competitive protein binding can be carried out with comparable procedures as heretofore employed; however, the sample, e.g., serum, is treated with tetrahydrofuran instead of alkanols or alkanol-acetone mixtures. The tetrahydrofuran is mixed with the serum sample in any convenient fashion without any extraordinary care in the addition of mixing steps. Mixing can be carried out rapidly using automatic agitating equipment or ultrasonic probe, thus eliminating the need for manual dexterity and skill formerly required for gentle, careful stirring with wire loops or the like. In addition, thyroxine recoveries averaging percent can be achieved on a routine basis, while using a volume of serum which is much less than formerly required.

In a convenient procedure for carrying out the process of the invention, a to 500 microliter sample of serum is mixed with from about 4 to about 9 times its volume of tetrahydrofuran in any order or fashion. The tetrahydrofuran and serum are vigorously mixed for a shortpredetermined period of time, after which protein is removed by centrifugation. The vigorous mixing can be carried out by shaking, vibrating or stirring at a rapid rate.

In an exemplary procedure, 0.20 milliliter of serum is pipetted into a tube, and 0.80 milliliter of tetrahydrofuran is added thereto using an automatic pipet. The contents of the tube are mixed by vigorous agitation using a finger-type mixer (such as that sold under the name Turbomixer) for 10 seconds or an ultra-sonic probe for 2-3 seconds. Protein is separated by centrifugation at 1,000 r.p.m. for 1 minute.

As in the known procedures described by Murphy, U. S. Pat. No. 3,414,383, an aliquot of the supernatant liquid (0.2 to 0.3 milliliters) is then removed and dried, preferably under a nitrogen atmosphere at a temperature of 45 C. A series of thyroxine standard solutions are similarly dried. The dried sample and standards are mixed with a predetermined amount, e.g., 1 milliliter, of radioactive-labeled thyroxine I-TBG solution. The labeled thyroxine solution preferably comprises 1- labeled thyroxine and 0.5 percent late pregnancy serum (as a source of thyroxine binding globulin) in aqueous barbital buffer (pl-I 8.6, ionic strength 0.075). The preferred radioactive-labeled thyroxine solution is prepared, for example, by dissolving a small amount of I-labeled thyroxine (50-70 microcuries) in 5 mil liliters of late pregnancy serum (serum from human females in the last trimester of pregnancy) plus 80 milliliters of barbital buffer and incubating the solutions at 45C. for 10 minutes. The solution is diluted to a final volume of one liter with the barbital buffer. After dis- I ,solving the serum extract by mixing and incubation at 45C. for a predetermined equilibration time (such as 8 to 30 minutes) the solutions are cooled-to a temperature of about 4C. for 10 or more minutes. Sufficient ion exchange resin (such as about 40 milligrams of Amberlite CG 400, 100-200 mesh resin, in the chloride form) is added to'remove labeledl unbound thyroxine. The mixing of the slurry of resin and treated sample or standard is accomplished on a rotary mixer for at least 2 to 5, to 10 or moreminutes at 4C. The exact mixing time is not critical. While standing at 4C., little or no change in results has been observed with contact times from ten to over sixtyminutes, using ion exchange resin and radioactive labeled thyroxine and 0.5 percent late pregnancy serum in barbital buffer.

After holding the resin mixture and allowing the resin to settle, an aliquot of the supernatant solution is removed, placed in a counting cuvet, and counted, using a scintillation counter. Standard curves are then prepared from the results obtained with the, standard solutions. The thyroxine content of the sample is then determined from the standard curve in accordance with the known procedures.

The improved procedure provides excellent results, and gives 95 percent recoveries of thyroxine. Addition step using tetrahydrofuran requires no great degree of manual dexterity or skill, and can be easily adapted to permit the use of automatic pipets and a mechanical or ultra-sonic agitation apparatus. Since the process of the invention permits the use of smaller sample volumes, duplicate or triplicate samples can be employed if desired, without increasing the volume of initial sample required.

I claim:

1. In a method of determining thyroxine in biological fluids by the competitive protein-binding technique comprising separating thyroxine and precipitating proteins from a biological fluid; equilibrating separated thyroxine with a buffered solution of thyroxine-binding-globulin (TBG) and radioactive-labeled-thyroxine, separating unbound thyroxine from the buffered solution by contacting the solution with an anion exchange resin, and counting the labeled thyroxine remaining bound to theTBG with a scintillation counter, the improvement whereinz-thyroxine is separated and protein precipitated from the biological fluid by mixing the biological fluid with tetrahydrofuran.

2. The method of claim 1 wherein the biological fluid and tetrahydrofuran are mixed together by vigorous agitation.

3. The method of claim 2 wherein said mixing is carried out by adding tetrahydrofuran to from about 200 to about 500 microliters of serum, the tetrahydrofuran being employed in an amount of from about 4 to about 9 parts (by volume) per part (by volume) of serum; and thereafter vigorously agitating the mixture for a predetermined period of time.

4. The method of claim 1 wherein the thyroxinebinding-globulin solution employed in the equilibration step is a solution of radioactive-labeled-thyroxine and late pregnancy serum in an aqueous barbital buffer solution.

5. The method of claim 4 wherein said solution has a pH of about 8.6, an ionic strength of-about 0.075 and contains about 0.5 percent of late pregnancy serum. 

2. The method of claim 1 wherein the biological fluid and tetrahydrofuran are mixed together by vigorous agitation.
 3. The method of claim 2 wherein said mixing is carried out by adding tetrahydrofuran to from about 200 to about 500 microliters of serum, the tetrahydrofuran being employed in an amount of from about 4 to about 9 parts (by volume) per part (by volume) of serum; and thereafter vigorously agitating the mixture for a predetermined period of time.
 4. The method of claim 1 wherein the thyroxine-binding-globulin solution employed in the equilibration step is a solution of radioactive-labeled-thyroxine and late pregnancy serum in an aqueous barbital buffer solution.
 5. The method of claim 4 wherein said solution has a pH of about 8.6, an ionic strength of about 0.075 and contains about 0.5 percent of late pregnancy serum. 